The invention relates to an in vitro method of recognizing and diagnosing acute coronary syndroms, especially an acute myocardial infarction (AMI) in humans. Acute coronary syndroms comprise the syndroms of acute myocardial infarction and instable angina pectoris which are defined, respectively, by the WHO classification of AMI and the Braunwald classification of instable angina pectoris (Gillum R. F. et al. 1984, Am Hart J 108: 150-158; Braunwald E. et al. 1994, Circulation 90: 613-622). Acute coronary syndroms are a complex of diseases which occur frequently and often threaten life. An early, certain diagnosis and therapy may be decisive for the patient""s survival. That is true particularly of an acute myocardial infarction in which case delayed proper diagnosis and late therapy may have serious consequences for the patient. There are such diagnostic measures of myocardial infarctions as the electrocardiogram and the determination of various laboratory markers. With a majority of patients, the sensitivity of the electrocardiogram and of the known laboratory markers is insufficient for diagnosis in the early phase of a myocardial infarction. As regards changes typical of an infarction reflected in the ECG (ST deflections), the sensitivity is 46% (Rude, R. E. et al. 1983, Am J Cardio 52: 936-942). The sensitivity of the laboratory markers creatine kinase (CK) activity, CK-MB activity, CK-MB mass, CK-MB isoforms, of myoglobin, and cardial troponins lies between 11-29% during the first two hours after the onset of pain (Mair, J. et al. Mitteilungen der Deutschen Gesellschaft fxc3xcr klinische Chemie 25: 1-6). The limited diagnostic usefulness of the known methods during the early phase of an acute myocardial infarction causes a number of clinical problems, such as the risk of erroneous diagnoses, carrying out treatments not indicated, and delaying life-saving therapies.
In the case of instable angina pectoris, patients at risk can be recognized with relative certainty by determining the cardial troponins. Due to the slow release kinetics, however, here too false negative findings are arrived at during the early phase. That is a problem because the prognosis of these patients may be about as bad as that of AMI patients and they need quick purposive antiischemic therapy. Considerable demand thus exists for methods which permit reliable, early recognition of acute coronary syndroms, particularly of an acute myocardial infarction.
It is, therefore, the object of the invention to indicate a method which allows early recognition of acute coronary syndroms, thus improving diagnosis and therapy of the stricken patients.
This object is met by a method comprising the features recited in claim 1. With the method according to the invention of recognizing and diagonising acute coronary syndroms, especially an acute myocardial infarction, the content of choline, choline and/or trimethyl ammonium derivatives selected from the group comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline is determined and evaluated in body fluids or component parts of the body withdrawn from a patient.
Choline, choline and/or trimethyl ammonium derivatives selected from the group comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline are molecules of the lipid meta-bolism and will be referred below in summary as xe2x80x9cCCTDxe2x80x9d. CCTD have the following chemical formulae: 
R1 and R2 represent certain chemical substituents characterizing a group of substances comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline. Plasmalogens and lysoplasmenyl choline contain an alkenyl group in these molecule parts.
Formula 1 indicates that choline represents [2-hydroxyethyl] trimethyl ammonium together with its counter ion. Formula 2 gives the general chemical formula of choline derivatives, and formula 3 presents the general formula of trimethyl ammonium derivatives, such as phosphoryl choline, as well as of plasmalogens, such as plasmenyl choline and lysoplasmenyl. The negative charge may be in the same molecule or in a counter ion. The majority of the CCTD either are component parts of phospholipids which in turn represent elements of biological membranes or are closely related to the phospholipid metabolism. Cardiac muscle cells are particularly rich in plasmenyl cholines which represent choline-containing phospholipids having a characteristic alkenyl group in the molecule. Plasmenyl cholines, furthermore, are component parts of membranes of the mitochondria which account for a considerable part of the myocardial mass. Results obtained by the applicants show that the activation of various myocardial phospholipases in the early phase of the acute myocardial infarction as well as disturbances of the lipid metabolism upon severe myocardial ischemia bring about a distinct release of choline, choline derivatives, and of the chemically related trimethyl ammonium derivatives, such as phosphoryl choline, plasmalogens, or lysoplasmenyl choline, thereby provoking an increase in concentration of CCTD in certain body fluids and component parts of the body. The extent of participation of non-myocyte elements, including endothelium cells and smooth vascular muscle cells is not known. The chemical relationship existing between choline, choline derivatives, and trimethyl ammonium derivatives selected from the group comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline as well as their similar behavior in pathophysiological processes, i. e. the very early release from the heart by ischemic membrane destruction are the reason for looking at them together, as CCTD.
Possible differences between the various CCTD stand back from the overwhelming common characteristic of very quick myocardial release in the event of acute coronary syndroms. This is all the more so considering the fact that the common characteristic mentioned is decisive for the essential feature of the method according to the invention in early diagnosis during the first few hours following the onset of pain.
Diagnostic utilization of the release of choline, choline derivatives, and trimethyl ammonium derivatives selected from the group comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline for recognition of acute coronary syndroms and acute myocardial infarction in humans has not been described as yet. The only thing known so far is that in experimental test arrangements in the early phase of myocardial ischemia a rise is observed of lysophosphoglycerides (e.g. lysophosphatidyl choline) in the myocardium and in venous as well as lymphatic effluvia (Corr, P. B. et al. 1987, J Mol Cell Cardiol 19: 34-53; Snyder, D. W. 1981, Am J Physiol 241: H700-H707; Akita, H. et al. 1986, J Clin Invest 78: 271-280). None of the publications applied a method of evaluating the content of choline, choline derivatives, and trimethyl ammonium derivatives selected from the group comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline in body fluids for diagnosing an acute coronary syndrom or an acute myocardial infarction in humans. The background of the publications cited, rather, is the observation that lysophosphatidyl choline apparently has proarrhythmic effects and that drugs having an anti-lysophosphatidyl choline effect possibly might be useful remedies.
The invention relates to a method of determining choline, choline and/or trimethyl ammonium derivatives, such as phosphoryl choline, plasmalogens, and lysoplasmenyl choline and/or certain reaction products for early diagnosis of acute coronary syndroms. A distinction must be made between non-inventive diacyl phosphatidyl cholines and inventive plasmenyl cholines. In the same manner, the non-inventive lysophosphatidyl cholines having an acyl group in the molecule must be distinguished from the inventive lysoplasmenyl cholines which have an alkenyl group in the molecule. Unspecific methods of determination which do not distinguish the substances according to the invention from other phospholipids or their components do not comprise the features of the method according to the invention which are essential for the early diagnosis of acute coronary syndroms. Therefore, they cannot be equated with the method.
Unspecific methods of determination of the whole group of plasma, serum, or whole blood phospholipids or lysophosphatidyl cholines, for instance, do not show the characteristics of the method according to the invention in the early diagnosis of acute coronary syndroms. These unspecific methods relating to phospholipids, as described in prior publications, essentially measure hepatic phospholipids in blood. And these hepatic phospholipids, as a matter of fact, do not contain the plasmenyl cholines and lysoplasmenyl cholines according to the invention. Instead they contain, quite predominantly, choline phospolipids without an alkenyl group, e.g. diacyl phosphatidyl choline. In view of the fact that the substances released by the heart into the blood in the event of acute coronary syndroms are not measured specifically by the non-inventive methods of determination, the methods in question do not comprise the features of the method according to the invention. In case of a myocardial infarction the non-inventive methods of determining phospholipids thus provide measurements either of no significant changes at all or even of reduced concentrations. This demonstrates that the methods in question do not detect the release of substances from the heart and a corresponding augmentation of the concentration thereof in blood. When determining the whole group of lysolecithins, for example, upon an acute myocardial infarction, often a reduction in concentration is measured because, in the unspecific acute-phase reaction, the formation of various acyl lysophosphatidyl cholines is reduced due to a decline in activity of the lecithin cholesterol acyl transferase (LCAT). Consequently measurements made with unspecific methods of determination of phospholipids often lead to contrary results and to conclusions which are not true of the method according to the invention.
The method according to the invention may be practiced with different analytical techniques, provided the substances according to the invention can be determined with sufficient specificity.
For instance, NMR spectroscopy may be applied upon preanalytical centrifugal ultrafiltration of the sample and removal of protein-bound diacyl phosphatidyl cholines which are not readily soluble. It is likewise possible to employ chromatographic, biochemical, or immunological methods of determination or other techniques. The analytical procedure may be conducted along the lines of methods of determination which have been published, for example, biochemical or enzymatic methods (Takayama, M. et al. 1977, Clin Chim Acta 79: 93-98), high performance liquid chromatography (HPLC) (Brouwers, J. F. H. et al. 1998, J Lipid Res 39: 344-353; Potter, P. et al. 1983, J Neurochem 41: 188-94) and gas chromatography and/or mass spectrometry (Myher, J. J. et al. 1989, Lipids 24: 396-407; Pomfret, A. et al. 1989, Anal Biochem 180:85) or immunological methods (Smal, M. A. et al. 1991, Lipids 26: 1130-1135; Baldo, B. A. et al. 1991, Lipids 26: 1136-1139). A survey of the analytics of phospholipids was published by Olsson (Olsson, N. U. et al.). We refer to these publications.
With biochemical methods according to the invention, for example, reagents (e.g. enzymes) are added to bring about chemical reactions or reaction products which then will permit detection and measurement of the whole group, a subgroup, or a subspecies of the CCTD (cf. examples of use).
With immunological methods according to the invention, for example, immunological reagents (antibodies) are used, usually together with other chemical and/or immunological reagents, to provoke reactions or yield reaction products which then will permit detection and measurement of the whole group, a subgroup, or a subspecies of the CCTD (cf. examples of use).
With chromatographic methods according to the invention, for example, upon preparation of the sample, a distribution of substances is effected between stationary and mobile phases so as to provide corresponding qualitative and quantitativeÿ data of CCTD (cf. examples of use).
It should be kept in mind with each analytical method of CCTD and their reaction products that some CCTD may form single layers, double layers, membranes, micelles, and/or vesicles and that these phenomena may be significant for the analyses.
Depending on the method of determination chosen, quantitative statements will be allowed to be made about certain individual groups or the entire group of substances analysed according to the invention. The prior publications mentioned are incorporated in the instant application by reference.
Certain activated phospholipases are significant in the cardial release of CCTD in the context of ischemic membrane destruction of cardiac muscle cells. These phospholipases cleave phospholipids so that, apart from CCTD, also simple reaction products result which, although not carrying a trimethyl ammonium group, still are released in the same way. Since these simple reaction products of CCTD are part of a common release mechanism the method can be carried out also by determining such simple reaction products. In addition to phospholipase A2 which, among others, contributes to the release of the lysoplasmenyl choline mentioned, further important enzymes of this group are phospholipases C and D attacking the ester bond between the hydroxy group at the 3-sn-C atom of glycerol and phosphoryl choline (phospholipase C) or the bond between the phosphoryl group of the 1,2 substituted glycerol phosphate and the choline which is esterified with the phosphoryl group (phospholipase D). Reaction products resulting from the activity of these two phospholipases acting on the plasmalogens thus are 1-O-alk-1xe2x80x2-enyl-2 substituted glycerol (from phospholipase C) and 1-O-alk-1xe2x80x2-enyl-2 substituted glycerol phosphate (from phospholipase D), respectively. These reaction products are set free together with choline and phosphoryl choline, respectively, when the phospholipases mentioned exert their influence on plasmenyl choline. Consequently, the method according to the invention of an early diagnosis of acute coronary syndroms can be carried out by determining choline, choline and/or trimethyl ammonium derivatives selected from the group comprising phosphoryl choline, plasmalogens, and lysoplasmenyl choline, and/or the reaction products thereof selected from the group comprising 1-O-alk-1xe2x80x2-enyl-2 substituted glycerol and 1-O-alk-1xe2x80x2-enyl-2 substituted glycerol phosphate.
The increase of CCTD and their reaction products may be caused by the following further processes, in addition to the increased phospholipid activity: reduced decomposition and/or increased synthesis of CCTD and their reaction products (with corresponding changes of the responsible enzymes) and release from cellular compartments and membranes due to other factors (mechanical factors, cell swelling, and other phenomena of myocytic lesion).